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Open AccessArticle

Effects of Heterologous tRNA Modifications on the Production of Proteins Containing Noncanonical Amino Acids

1
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA
2
Department of Chemistry, Yale University, New Haven, CT 06520, USA
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Academic Editor: Gary Chinga Carrasco
Bioengineering 2018, 5(1), 11; https://doi.org/10.3390/bioengineering5010011
Received: 19 December 2017 / Revised: 31 January 2018 / Accepted: 31 January 2018 / Published: 2 February 2018
(This article belongs to the Special Issue Novel Biocomposite Engineering and Bio-Applications)
Synthesis of proteins with noncanonical amino acids (ncAAs) enables the creation of protein-based biomaterials with diverse new chemical properties that may be attractive for material science. Current methods for large-scale production of ncAA-containing proteins, frequently carried out in Escherichia coli, involve the use of orthogonal aminoacyl-tRNA synthetases (o-aaRSs) and tRNAs (o-tRNAs). Although o-tRNAs are designed to be orthogonal to endogenous aaRSs, their orthogonality to the components of the E. coli metabolism remains largely unexplored. We systematically investigated how the E. coli tRNA modification machinery affects the efficiency and orthogonality of o-tRNASep used for production of proteins with the ncAA O-phosphoserine (Sep). The incorporation of Sep into a green fluorescent protein (GFP) in 42 E. coli strains carrying deletions of single tRNA modification genes identified several genes that affect the o-tRNA activity. Deletion of cysteine desulfurase (iscS) increased the yield of Sep-containing GFP more than eightfold, while overexpression of dimethylallyltransferase MiaA and pseudouridine synthase TruB improved the specificity of Sep incorporation. These results highlight the importance of tRNA modifications for the biosynthesis of proteins containing ncAAs, and provide a novel framework for optimization of o-tRNAs. View Full-Text
Keywords: noncanonical amino acids; genetic code expansion; protein translation; tRNA; aminoacyl-tRNA synthetases; posttranscriptional modifications; phosphoserine noncanonical amino acids; genetic code expansion; protein translation; tRNA; aminoacyl-tRNA synthetases; posttranscriptional modifications; phosphoserine
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MDPI and ACS Style

Crnković, A.; Vargas-Rodriguez, O.; Merkuryev, A.; Söll, D. Effects of Heterologous tRNA Modifications on the Production of Proteins Containing Noncanonical Amino Acids. Bioengineering 2018, 5, 11.

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